RESUMEN
The application of ketogenic diet (KD) (high fat/low carbohydrate/adequate protein) as an auxiliary cancer therapy is a field of growing attention. KD provides sufficient energy supply for healthy cells, while possibly impairing energy production in highly glycolytic tumor cells. Moreover, KD regulates insulin and tumor related growth factors (like insulin growth factor-1, IGF-1). In order to provide molecular evidence for the proposed additional inhibition of tumor growth when combining chemotherapy with KD, we applied untargeted quantitative metabolome analysis on a spontaneous breast cancer xenograft mouse model, using MDA-MB-468 cells. Healthy mice and mice bearing breast cancer xenografts and receiving cyclophosphamide chemotherapy were compared after treatment with control diet and KD. Metabolomic profiling was performed on plasma samples, applying high-performance liquid chromatography coupled to tandem mass spectrometry. Statistical analysis revealed metabolic fingerprints comprising numerous significantly regulated features in the group of mice bearing breast cancer. This fingerprint disappeared after treatment with KD, resulting in recovery to the metabolic status observed in healthy mice receiving control diet. Moreover, amino acid metabolism as well as fatty acid transport were found to be affected by both the tumor and the applied KD. Our results provide clear evidence of a significant molecular effect of adjuvant KD in the context of tumor growth inhibition and suggest additional mechanisms of tumor suppression beyond the proposed constrain in energy supply of tumor cells.
Asunto(s)
Dieta Cetogénica , Metaboloma , Metabolómica , Neoplasias/metabolismo , Acetilación , Aminoácidos/biosíntesis , Aminoácidos/metabolismo , Animales , Línea Celular Tumoral , Cromatografía Líquida de Alta Presión , Modelos Animales de Enfermedad , Ácidos Grasos/metabolismo , Xenoinjertos , Humanos , Metabolómica/métodos , Ratones , Neoplasias/patología , Espectrometría de Masas en TándemRESUMEN
The present contribution illustrates the utilization of a chiral × chiral two-dimensional liquid chromatography (2DLC) setup with tert-butylcarbamoyl quinine chiral stationary phase (CSP) in the first dimension (1D) and tert-butylcarbamoyl quinidine CSP in the second dimension (2D) to analyze FMOC-derivatized d and l amino acids from peptide hydrolysates. Hereby, in the 1D and 2D chiral separation dimensions factors such as selector and immobilization chemistry of the CSPs, mobile phase, temperature, column hardware dimensions, stationary phase supports, particle type and packing were identical. Orthogonality between 1D and 2D CSPs was solely based on their stereochemistry, i.e. their opposite configurations in two chiral centers of the selector molecules, which results in inversion of enantiomer elution orders in the two dimensions. Using Coreshell CSPs for fast chromatography allowed 2D-flow rates which were 60 times faster than the 1D-flow rates to enable online comprehensive two-dimensional chromatography (LC × LC). Due to very similar chemoselectivity, yet opposite elution orders of corresponding enantiomers in 1D and 2D, characteristic 2D-elution patterns for achiral and chiral components can be generated. Peaks of achiral components and impurities are lined up on the diagonal line in the 2D separation space (contour plot) and thereby removed from the chromatographic space of the target enantiomers avoiding overlaps with potential interferences. Corresponding enantiomers provide cross peaks on the 2D chromatogram. Moreover, enantioselectivity of both single CSPs is combined to result in an enhanced overall 2D enantioselectivity. The concept is illustrated for the therapeutic peptides gramicidin and bacitracin. Since all amino acids give a consistent elution order as FMOC-derivatives, all enantiomers of the same configuration are either above or below the diagonal line allowing straightforward imaging of the configuration of the amino acids in peptides by the 2D chromatogram.
Asunto(s)
Aminoácidos/química , Bacitracina/química , Cromatografía Liquida/métodos , Gramicidina/química , Hidrólisis , EstereoisomerismoRESUMEN
Untargeted metabolomics has the potential to improve the predictivity of in vitro toxicity models and therefore may aid the replacement of expensive and laborious animal models. Here we describe a long term repeat dose nephrotoxicity study conducted on the human renal proximal tubular epithelial cell line, RPTEC/TERT1, treated with 10 and 35 µmol·liter(-1) of chloroacetaldehyde, a metabolite of the anti-cancer drug ifosfamide. Our study outlines the establishment of an automated and easy to use untargeted metabolomics workflow for HPLC-high resolution mass spectrometry data. Automated data analysis workflows based on open source software (OpenMS, KNIME) enabled a comprehensive and reproducible analysis of the complex and voluminous metabolomics data produced by the profiling approach. Time- and concentration-dependent responses were clearly evident in the metabolomic profiles. To obtain a more comprehensive picture of the mode of action, transcriptomics and proteomics data were also integrated. For toxicity profiling of chloroacetaldehyde, 428 and 317 metabolite features were detectable in positive and negative modes, respectively, after stringent removal of chemical noise and unstable signals. Changes upon treatment were explored using principal component analysis, and statistically significant differences were identified using linear models for microarray assays. The analysis revealed toxic effects only for the treatment with 35 µmol·liter(-1) for 3 and 14 days. The most regulated metabolites were glutathione and metabolites related to the oxidative stress response of the cells. These findings are corroborated by proteomics and transcriptomics data, which show, among other things, an activation of the Nrf2 and ATF4 pathways.
Asunto(s)
Acetaldehído/análogos & derivados , Antineoplásicos/toxicidad , Nefronas/metabolismo , Acetaldehído/toxicidad , Línea Celular , Cromatografía Líquida de Alta Presión , Humanos , Metaboloma , Nefronas/efectos de los fármacos , Programas Informáticos , Espectrometría de Masas en TándemRESUMEN
BACKGROUND: The rapidly increasing number of engineered nanoparticles (NPs), and products containing NPs, raises concerns for human exposure and safety. With this increasing, and ever changing, catalogue of NPs it is becoming more difficult to adequately assess the toxic potential of new materials in a timely fashion. It is therefore important to develop methods which can provide high-throughput screening of biological responses. The use of omics technologies, including metabolomics, can play a vital role in this process by providing relatively fast, comprehensive, and cost-effective assessment of cellular responses. These techniques thus provide the opportunity to identify specific toxicity pathways and to generate hypotheses on how to reduce or abolish toxicity. RESULTS: We have used untargeted metabolome analysis to determine differentially expressed metabolites in human lung epithelial cells (A549) exposed to copper oxide nanoparticles (CuO NPs). Toxicity hypotheses were then generated based on the affected pathways, and critically tested using more conventional biochemical and cellular assays. CuO NPs induced regulation of metabolites involved in oxidative stress, hypertonic stress, and apoptosis. The involvement of oxidative stress was clarified more easily than apoptosis, which involved control experiments to confirm specific metabolites that could be used as standard markers for apoptosis; based on this we tentatively propose methylnicotinamide as a generic metabolic marker for apoptosis. CONCLUSIONS: Our findings are well aligned with the current literature on CuO NP toxicity. We thus believe that untargeted metabolomics profiling is a suitable tool for NP toxicity screening and hypothesis generation.
Asunto(s)
Metabolómica , Nanopartículas del Metal/toxicidad , Apoptosis , Biomarcadores/metabolismo , Línea Celular Tumoral , Glutatión Peroxidasa/metabolismo , Hemo-Oxigenasa 1/metabolismo , Humanos , Interleucina-8/metabolismo , Neoplasias Pulmonares/metabolismo , Neoplasias Pulmonares/patología , Nanopartículas del Metal/química , Microscopía Electrónica de Transmisión , Estrés Oxidativo , Glutatión Peroxidasa GPX1RESUMEN
Chemical cross-linking in combination with mass spectrometric analysis offers the potential to obtain low-resolution structural information from proteins and protein complexes. Identification of peptides connected by a cross-link provides direct evidence for the physical interaction of amino acid side chains, information that can be used for computational modeling purposes. Despite impressive advances that were made in recent years, the number of experimentally observed cross-links still falls below the number of possible contacts of cross-linkable side chains within the span of the cross-linker. Here, we propose two complementary experimental strategies to expand cross-linking data sets. First, enrichment of cross-linked peptides by size exclusion chromatography selects cross-linked peptides based on their higher molecular mass, thereby depleting the majority of unmodified peptides present in proteolytic digests of cross-linked samples. Second, we demonstrate that the use of proteases in addition to trypsin, such as Asp-N, can additionally boost the number of observable cross-linking sites. The benefits of both SEC enrichment and multiprotease digests are demonstrated on a set of model proteins and the improved workflow is applied to the characterization of the 20S proteasome from rabbit and Schizosaccharomyces pombe.
Asunto(s)
Cromatografía en Gel , Reactivos de Enlaces Cruzados/farmacología , Péptido Hidrolasas/metabolismo , Complejo de la Endopetidasa Proteasomal/metabolismo , Proteínas/metabolismo , Schizosaccharomyces/metabolismo , Animales , Bovinos , Cromatografía Liquida , Fragmentos de Péptidos/metabolismo , Conformación Proteica , Conejos , Espectrometría de Masas en Tándem , Tripsina/farmacologíaRESUMEN
Acetaldehyde is a strongly electrophilic compound that is endogenously produced as a first intermediate in oxidative ethanol metabolism. Its high reactivity towards biogenic nucleophiles has toxicity as a consequence. Acetaldehyde readily undergoes a non-enzymatic condensation reaction and consecutive ring formation with cysteine to form 2-methylthiazolidine-4-carboxylic acid (MTCA). For analytical purposes, N-acetylation of MTCA was required for stabilization and to enable its quantification by reversed-phase chromatography combined with electrospray ionization-tandem mass spectrometry. Qualitative screening of post mortem blood samples with negative blood alcohol concentration (BAC) mostly showed low basal levels of MTCA. In BAC-positive post mortem samples, but not in corresponding urine specimens, strongly increased levels were present. To estimate the association between ethanol consumption and the occurrence of MTCA in human blood, the time curves of BAC and MTCA concentration were determined after a single oral dose of 0.5 g ethanol per kilogram of body weight. The blood elimination kinetics of MTCA was slower than that of ethanol. The peak concentration of MTCA (12.6 mg L(-1)) was observed 4 h after ethanol intake (BAC 0.07) and MTCA was still detectable after 13 h. Although intermediary acetaldehyde scavenging by formation of MTCA is interesting from a toxicological point of view, lack of hydrolytic stability under physiological conditions may hamper the use of MTCA as a quantitative marker of acetaldehyde exposure, such as resulting from alcohol consumption.
Asunto(s)
Acetaldehído/química , Consumo de Bebidas Alcohólicas/sangre , Cisteína/química , Tiazoles/sangre , Acetaldehído/metabolismo , Adulto , Consumo de Bebidas Alcohólicas/efectos adversos , Consumo de Bebidas Alcohólicas/metabolismo , Autopsia , Cisteína/metabolismo , Etanol/efectos adversos , Etanol/metabolismo , Humanos , Masculino , Estructura Molecular , Tiazoles/químicaRESUMEN
Phosphorylated carbohydrates are important intracellular metabolites and thus of prime interest in metabolomics research. Complications in their analysis arise from the existence of structural isomers that do have similar fragmentation patterns in MS/MS and are hard to resolve chromatographically. Herein, we present selective methods for the liquid chromatographic separation of sugar phosphates, such as hexose and pentose phosphates, 2- and 3-phosphoglycerate, dihydroxyacetone phosphate and glyceraldehyde 3-phosphate, as well as glucosamine 1- and 6-phosphate utilizing mixed-mode chromatography with reversed-phase/weak anion-exchangers and a charged aerosol detector. The best results were obtained when the reversed-phase/weak anion-exchanger column was operated under hydrophilic interaction liquid chromatography elution conditions. The effects of various chromatographic parameters were examined and are discussed on the basis of a simple stoichiometric displacement model for explaining ion-exchange processes. Employed acidic conditions have led to the complete separation of α- and ß-anomers of glucose 6-phosphate at low temperature. The anomers coeluted in a single peak at elevated temperatures (>40°C) (peak coalescence), while at intermediate temperatures on-column interconversion with a plateau in-between resolved anomer peaks was observed with apparent reaction rate constants between 0.1 and 27.8×10(-4) s(-1). Dynamic HPLC under specified conditions enabled to investigate mutarotation of phosphorylated carbohydrates, their interconversion kinetics, and energy barriers for interconversion. A complex mixture of six hexose phosphate structural isomers could be resolved almost completely.
Asunto(s)
Cromatografía por Intercambio Iónico/métodos , Metabolómica/métodos , Fosfatos de Azúcar/química , Fosfatos de Azúcar/aislamiento & purificación , Cromatografía Líquida de Alta Presión/métodos , Concentración de Iones de Hidrógeno , Interacciones Hidrofóbicas e Hidrofílicas , Isomerismo , Estructura Molecular , FosforilaciónRESUMEN
Plants synthesize a number of different oligomeric or polymeric sugars containing galactose. During growth and development some of these carbohydrates are metabolized or remodeled releasing galactose as a breakdown product. All plants have established recycling pathways for such sugars, for which they seem to have a limited capacity to cope with. Exceeding these limits results in sugar toxicity, which is observed already at concentrations as low as 1 mmol·l-1 for galactose. The mechanism of galactose toxicity is poorly understood but it seems plausible that the enzymes involved in carbohydrate metabolism also might be the targets responsible for the adverse effects. Data from yeast and bacteria suggests that the enzyme phosphoglucomutase (PGM) is inhibited by galactose-1-phosphate. To test this hypothesis for plants we expressed recombinant cytosolic PGM3 from Arabidopsis in E. coli. Intriguingly, the enzyme was not inhibited by galactose-1-phosphate at physiological concentrations. Furthermore, PGM3 did not convert galactose-1-phosphate to galactose-6-phosphate, which was suggested as the inhibitory mode of action in yeast. In addition, metabolite levels in Arabidopsis roots were analyzed for their galactose-1-phosphate concentration by means of GC-MS. Seedlings grown on MS-media with sucrose contained less than 10 nmol·g FW-1 of galactose-1-phosphate. However, seedlings from plates, in which the sucrose was replaced by galactose, showed a strong increase of Gal-1-P to levels of up to 200 nmol·g FW-1.
RESUMEN
Novel chiral stationary phases (CSPs) based on zwitterionic Cinchona alkaloid-type low-molecular mass chiral selectors (SOs), as they have been reported recently, were investigated in HPLC towards effects on their chromatographic behavior by mobile phase composition. Mobile phase characteristics like acid-to-base ratio and type of acidic and basic additives as well as effect of type of bulk solvents in nonaqueous polar organic and aqueous reversed-phase (RP) eluent systems were varied in order to illustrate the variability and applicability of zwitterionic CSPs with regard to mobile phase aspects. Chiral SOs of the five zwitterionic CSPs investigated herein contained weak and strong cation-exchange (WCX, SCX) sites at C9- and C6'-positions of the Cinchona alkaloid scaffold which itself accommodated the weak anion-exchange (WAX) site. The study focused on zwitterion-exchange (ZX) operational mode and chiral amino acids as target analytes. Besides, also the anion-exchange (AX) mode for chiral N-blocked amino acid analytes was considered, because of the intramolecular counterion (IMCI) property available in AX mode. Overall, most general and successful conditions in ZX mode were found to be weakly acidic methanolic mobile phases. In aqueous eluents RP contributions to retention came into play but only at low organic modifier content because of the highly polar character of zwitterionic analytes. At higher acetonitrile content, HILIC-related retention phenomena were observed. When using weakly basic eluent system in AX mode remarkably fast enantiomer separations involving exclusion phenomena were possible with one enantiomer eluting before and the other after void volume.
Asunto(s)
Cromatografía por Intercambio Iónico/métodos , Modelos Químicos , Quinina/química , Acetonitrilos , Aminoácidos/química , Concentración de Iones de Hidrógeno , Metanol , Concentración Osmolar , EstereoisomerismoRESUMEN
The concept of recently introduced Cinchona alkaloid-type zwitterionic chiral stationary phases (CSPs) is based on fusing key cation- and anion-exchange (CX, AX) moieties in one single low-molecular mass chiral selector (SO) with the resulting CSPs allowing enantiomer separations of a wide range of chiral ionizable analytes comprising acids, bases, and zwitterionic compounds. Herein, we report principal, systematic investigations of the ion-exchange-type retention mechanisms available with the novel zwitterionic CSPs in nonaqueous polar organic mode. Typical CX and AX processes, corresponding to the parent single ion exchangers, are confirmed also for zwitterionic CSPs. Also the mechanism leading to recognition and retention of zwitterions was found to be ion exchange mediated in a zwitterion-exchange (ZX) mode. In both AX and CX modes the additional ionizable group within the SO besides the site responsible for the respective ion-exchange process could be characterized as an intramolecular counterion (IMCI) that effectively participates in the ion-exchange equilibria and thus, contributes to solute elution. In the ZX mode both oppositely charged groups of the zwitterionic SO were found not only to be the sites for simultaneous ion pairing with the analyte but also functioned as IMCIs at the same time. The main practical consequences of the IMCI feature were significant reduction of the amounts and even elimination of acidic and basic additives required in the eluent systems to afford analyte elution while still providing faster analysis than the parent single ion-exchanger-type CSPs. The set of ten structurally different zwitterionic CSPs employed in this study facilitated the establishment of correlations between chromatographic behavior of the CSPs with particular SO elements, thereby supporting the understanding of the working principles of these novel packing materials on a molecular level.
Asunto(s)
Cromatografía por Intercambio Iónico/métodos , Modelos Químicos , Quinina/química , Aminoácidos/química , Concentración de Iones de Hidrógeno , EstereoisomerismoRESUMEN
The occurrence of d-amino acids (D-AAs) in higher-developed organisms in their free form, and within peptides and proteins, has been investigated with an increasing number of studies. Often the inversion of the stereochemical configuration of an individual amino acid drastically changes its biological activity. Alongside Asn and Asp, Ser is most prone to racemization within peptides. Specific enzymes catalyzing D-Ser generation and breakdown have been described. Hence, the applicability of enantioselective ZWIX(+)(®) and ZWIX(-)(®) chiral stationary phases (CSPs) to peptide separations was assessed and a set of 14 pairs of diastereomeric and enantiomeric Ser and Thr containing di-, tri- and tetra-peptides was chromatographically separated without prior hydrolysis to the individual amino acids. To a certain extent, RP chromatography also enabled the separation of peptide diastereomers. The ZWIX CSPs delivered chromatographic selectivities between 1.04 and 7.23, allowing a change of elution order by switching between the ZWIX(+) and the ZWIX(-) CSP. Coupling these highly selective chromatographic columns with an LTQ-Orbitrap XL™ mass spectrometer and performing high resolution MS(2) measurements enabled us to investigate mechanistic aspects of chemically induced racemization of Ser embedded in short peptides. As reaction medium an alkaline aqueous solution (pH 12.3) was selected. Proton/deuterium exchange experiments provided evidence of a fast Cα proton exchange with simultaneous racemization. Additionally, (18)O/(16)O exchange allowed the identification of an alternative, and somewhat retarded racemization via a reversible ß-elimination and reintroduction of water at the hydroxymethyl side chain of Ser. This involved the intermediate generation of the prochiral didehydro alanine unit.
Asunto(s)
Sistemas de Transporte de Aminoácidos Neutros/análisis , Fragmentos de Péptidos/análisis , Serina/análisis , Espectrometría de Masas en Tándem/métodos , Sistemas de Transporte de Aminoácidos Neutros/química , Cromatografía por Intercambio Iónico/métodos , Fragmentos de Péptidos/química , Serina/química , EstereoisomerismoRESUMEN
The determination of trace amounts of d-amino acids (d-AAs) even in tissue samples of higher developed animals, mammals and humans has opened a wide field of biological questions to be investigated. d-Ala, d-Asp and d-Ser have already been identified to exhibit key functions in cellular regulation processes [1-4]. The abundance of trace amounts of these and also of other d-AAs in various biological fluids and in tissue samples is still being investigated. We herein present a facile derivatization method for amino acids using 6-methoxyquinoline-4-carboxylic acid-succinimide ester (MQ-OSu) to yield the corresponding stable N-acyl-amino acids (MQ-AAs). Labeling with the MQ tag supports the enantioseparation of all 19 chiral proteinogenic amino acids on anion exchanger type chiral stationary phases, introduces fluorescence activity and particularly promotes sensitive electrospray tandem mass spectrometric detection. Limit of detection values (LOD) for MQ-l-Ala in water were 1.25 µmol L(-1) with fluorescence detection and 0.015 µmol L(-1) with MS in selected reaction monitoring (SRM) mode. The applicability of this method for the analysis of MQ-d-AAs in biological fluids has been demonstrated.
Asunto(s)
Aminoácidos/aislamiento & purificación , Cromatografía Líquida de Alta Presión/métodos , Quinolinas/química , Espectrometría de Fluorescencia/métodos , Espectrometría de Masas en Tándem/métodos , Aminoácidos/química , Aminoácidos/orina , Calibración , Línea Celular Tumoral , Humanos , EstereoisomerismoRESUMEN
D-Amino acid analysis in biological samples still poses a challenge to analytical chemists. In higher developed species trace amounts of d-amino acids have to be detected in vast excesses of the corresponding L-enantiomers. This method utilizes an easy-to-carry-out derivatization step on the amino group with an iron ferrocenyl propionate hydroxy succinimide ester followed by one-dimensional enantioselective anion exchange chromatography with cinchona alkaloid based chiral stationary phases (CSPs). MS detection is carried out in the highly sensitive SRM (selected reaction monitoring) mode, which allows a chemoselective differentiation of amino acid derivatives as well as their enantioselective separation in one step. Application of this method allows LOD (limits of detection) in the low µmol L(-1) range and baseline enantioseparation for all proteinogenic amino acids except for Pro, Arg and His. The D-enantiomers of isomeric Leu and Ile were separated chromatographically and pose an example for the complementary selectivities of LC and MS. A successful application of this procedure to unprocessed human urine indicated the eligibility to analyse biological samples.